Mycobacterial isocitrate lyase gene and uses thereof

ABSTRACT

The present invention provides a purified and isolated nucleic acid encoding mycobacterial isocitrate lyase, as well as mutated forms of the nucleic acid. Further provided are purified and isolated isocitrate lyase proteins and mutated isocitrate lyase proteins. Additionally, the present invention provides vectors which comprises nucleic acid sequences encoding mycobacterial isocitrate lyase and mutated forms of this nucleic acid, as well as host cells containing these vectors. Also provided is a mycobacterium containing one or more mutations in its isocitrate lyase gene. Further provided by the present invention are agents that inhibit the activity or expression of a mycobacterial lyase protein, a method of identifying these, and a method of producing them. Finally, the present invention also provides a method of identifying genes required for persistence of mycobacteria.

STATEMENT OF GOVERNMENT INTEREST

[0001] This invention was made with government support under NIH GrantNo. AI26170. As such, the government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0002] Pulmonary tuberculosis initiates with the inhalation andretention in the lung alveoli of a “droplet nucleus” containing from1-10 tubercle bacilli. Most cases of human tuberculosis originate from asingle primary lesion in the lung parenchyma; the number of bacilliinitiating an infection is therefore extremely small (Medlar). Patenttuberculous disease develops only after expansion of this initiallysmall bacillary population by replication within host macrophages. Inorder to grow, persist, and cause disease, tubercle bacilli must obtainnutrients from the parasitized host. Little is known, however, of themechanisms involved in nutrient acquisition by tubercle bacilli in vivo.Writing in 1976, Ratledge opined that “[T]he entire subject of in vivonutrition of bacteria when within the phagocytic cells of the host isprobably the largest single area of ignorance in the whole of ourknowledge concerning the physiology of the mycobacteria. Clearly this isa crucial area where knowledge should be sought as it is only byunderstanding the true behavior and requirements of the bacteria whengrowing in vivo that we shall learn how to prevent their multiplicationand, hopefully, how to cause their death” (Ratledge, 1976)Unfortunately, the intervening decades have marked little progress inthis area. With the advent of molecular genetic tools for themanipulation of the pathogenic mycobacteria, a genetic approach to thisproblem is now feasible.

[0003] In the infected host, M. tuberculosis bacilli replicate withinhost macrophages. Following phagocytosis, tubercle bacilli reside withinmodified phagosomes that apparently fuse with vacuoles derived from theendosomal compartment (Sturgill-Koszycki et al., 1996) but that fail toacidify fully or to fuse with lysosomes (reviewed in Clemens, 1996). Asan intracellular parasite, M. tuberculosis would potentially have accessto a variety of nutrients that are abundant within the host cellcytoplasm (Wheeler and Ratledge, 1994). The enclosure of tuberclebacilli within tightly apposed membranous vacuoles (Moreira et al.,1997) might, however, limit access to cytoplasmic constituents. Thisidea was supported by the recent demonstration that a leuD auxotroph ofthe attenuated bacille Calmette-Guerin (BCG) strain of tubercle bacilluswas incapable of replicating in mice (McAdam et al., 1995) or incultured macrophages (Bange et al., 1996). Although M. tuberculosis isnot a nutritionally fastidious organism, bacillary growth does require acarbon substrate(s) to provide building blocks for biosyntheticreactions and energy for metabolism. In vitro, M. tuberculosis iscapable of utilizing a wide range of carbon substrates, includingcarbohydrates, amino acids, and C2 carbon sources such as acetate andfatty acids (Wayne, 1994). It is not known which of these substrates areavailable to M. tuberculosis replicating within the confines of thephagosomal compartment.

[0004] Extensive biochemical studies have been made of tubercle bacilliisolated directly from the lungs of chronically infected mice (reviewedin Segal 1984). Using manometry, Segal and Bloch (1956) showed thatthese “in vivo grown” bacilli displayed a vigorous respiratory responseto fatty acids but failed to respond to a variety of other substrates.In contrast, respiration of tubercle bacilli grown in vitro was readilystimulated by both glucose and glycerol, which are the preferredsubstrates for in vitro cultivation of tubercle bacilli. Theseobservations suggested that tubercle bacilli in vivo may be adapted toutilization of fatty acids and may repress pathways for utilization ofother carbon sources. Later studies revealed that in vivo grown bacilliretained the ability to oxidize ¹⁴C-glucose to ¹⁴C-CO₂, but thataddition of exogenous glucose suppressed the respiration of endogenoussubstrates presumably including fatty acids (Artman and Bekierkunst,1960).

[0005] Two specialized pathways are required for utilization of fattyacids as sole carbon source. The b-oxidation pathway catalyzes thebreakdown of fatty acids to assimilable acetyl CoA units, which arefurther oxidized via the Krebs cycle (Clark and Cronan, 1996). Theglyoxylate shunt is an anaplerotic pathway for replenishment ofessential Krebs cycle intermediates consumed by biosynthetic pathwaysduring growth on C₂ carbon sources such as fatty acids and acetate(Cronan and LaPorte, 1996). This anaplerotic function is subsumed bypyruvate carboxylase when cells are grown on carbohydrates. Wheeler andRatledge (1988) found that in vivo grown mycobacteria readily oxidized[¹⁴C]-palmitate to [¹⁴C]-CO2, implying that the enzymes required forb-oxidation of fatty acids were expressed in vivo. (In fact, evolutionof [¹⁴C]-CO2 from [¹⁴C]-palmitate is the basis of the widely used“BACTEC” system for detection of M. tuberculosis in clinical specimens[Heifets and Good, 1994].) In addition, these authors demonstratedexpression of both enzymes of the glyoxylate shunt (malate synthase andisocitrate lyase) by in vivo grown mycobacteria. In Escherichia coli,expression of the enzymes of the b-oxidation pathway and of theglyoxylate shunt is under transcriptional control: transcription isrepressed during growth on carbohydrates and is induced during growth onfatty acids. Although these enzymes and their regulation have beencharacterized only partially in mycobacteria, their expression by invivo grown bacilli suggests that fatty acids may be utilized in vivo. Ifso, then the b-oxidation pathway and the glyoxylate shunt may beessential for in vivo growth or persistence of tubercle bacilli.

SUMMARY OF THE INVENTION

[0006] The present invention provides a purified and isolated nucleicacid encoding mycobacterial isocitrate lyase. The present inventionspecifically provides for nucleic acid sequences encoding mycobacterialisocitrate lyase that are obtained from M. tuburculosis and M.smegmatis. Also provided by the present invention are mutated nucleicacid sequences encoding mycobacterial isocitrate lyase.

[0007] Additionally, the present invention provides vectors whichcomprises the nucleic acid sequences encoding mycobacterial isocitratelyase of the present invention, and vectors which comprises the mutatednucleic acid sequences encoding mycobacterial isocitrate lyase of thepresent invention, as well as host cells containing these vectors.

[0008] Further provided by the present invention is an agent thatinhibits the activity or expression of a mycobacterial lyase protein, amethod of identifying agents that inhibit the activity or expression ofa mycobacterial lyase protein, and a method of producing the agents.

[0009] Finally, the present invention provides a method of identifyinggenes required for persistence of mycobacteria.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1A sets forth the nucleotide sequence of the M. tuburculosisisocitrate lyase gene. FIG. 1B sets forth the amino acid sequence of theM. tuburculosis isocitrate lyase gene.

[0011]FIG. 2A sets forth the nucleotide sequence of the M. smegmatisisocitrate lyase gene. FIG. 1B sets forth the amino acid sequence of theM. smegmatis isocitrate lyase gene.

[0012] FIGS. 3A-3H set forth the screening results of Ace mutants of M.smegmatis.

[0013]FIG. 4A sets forth a diagram indicating the position of the geneencoding 3-hydroxybutyryl-CoA dehydrogenase in relation to theisocitrate lyase gene in M. tuberculosis. FIG. 4B sets forth a diagramindicating the position of the gene encoding 3-hydroxybutyryl-CoAdehydrogenase in relation to the isocitrate lyase gene in M. smegmatis.

[0014]FIG. 5 sets forth an amino acid sequence comparison between the M.tuberculosis isocitrate lyase gene, the M. smegmatis isocitrate gene,and the isocitrate lyase gene from Rhodococcus fasciens.

[0015]FIG. 6A sets forth a diagram showing the targeted disruption ofthe isocitrate lyase gene in M. tuberculosis. FIG. 6B sets forth theresults of a Southern blot analysis of mutants generated by the targeteddisruption.

[0016]FIG. 7A sets forth a graph depicting the growth of an M.tuberculosis isocitrate lyase mutant. FIG. 7B sets forth a graphdepicting the percent survival of an M. tuberculosis isocitrate lyasemutant.

[0017]FIG. 8A sets forth a graph depicting the ability of an M.tuberculosis isocitrate lyase mutant to grow and persist in a mousemodel. FIG. 8B indicates that persistence of the M. tuberculosisisocitrate lyase mutant in infected mice was impaired.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides a purified and isolated nucleicacid encoding mycobacterial isocitrate lyase. As used herein, thenucleic acid may be genomic DNA, cDNA, or RNA. Due to the degeneracy ofthe genetic code, the nucleic acid of the present invention alsoincludes a multitude of nucleic acid substitutions which will encodeisocitrate lyase.

[0019] The present invention specifically provides for a nucleic acidencoding mycobacterial isocitrate lyase that is isolated fromMycobacteria tuburculosis. Preferably, the nucleic acid sequenceencoding M. tuburculosis isocitrate lyase encodes the amino acidsequence contained in FIG. 1. More preferably, the M. tuburculosisisocitrate lyase nucleic acid has the nucleotide sequence contained inFIG. 1. The present invention also provides for nucleic acid encodingmycobacterial isocitrate lyase that is isolated from Mycobacteriasmegmatis. Preferably, the nucleic acid sequence encoding M. smegmatisisocitrate lyase encodes the amino acid sequence contained in FIG. 2.More preferably, the M. smegmatis isocitrate lyase nucleic acid has thenucleotide sequence contained in FIG. 2.

[0020] Further provided by the present invention is a mutated nucleicacid sequence encoding mycobacterial isocitrate lyase. The mutatednucleic acid sequence encoding mycobacterial isocitrate lyase may beisolated from M. tuburculosis, M, smegmatis, M. avium, M. kansasii, M.zenopi, M. simiae, M. gastri, M. szulgai, M. gordonae, M. chelonea, M.leprae, M. bovis-BCG, M. intracellulare, M. habana, M. lufu, M. phlei,M. fortuitum, M. paratuburculosis and M. scrofulaceum. The mutation maybe generated in said nucleic acid using methods known to one of skill inthe art. Such methods of mutation include, but are not limited to,signature-tagged mutagenesis, transposon mutagenesis, targeted genedisruption, illegitimate recombination and chemical mutagenesis. In apreferred embodiment of the invention, the mutated nucleic acid encodingisocitrate lyase is M. tuburculosis nucleic acid. In a more preferredembodiment on the invention, the mutation in the M. tuburculosis nucleicacid encoding isocitrate lyase is generated by disruption. Disruption ofa nucleic acid encoding isocitrate lyase may be performed, for example,by allelic exchange. It is to be understood that the present inventionalso provides for nucleic acid sequences wherein any or all of the abovedescribed mutations coexist in the nucleic acid encoding mycobacterialisocitrate lyase in any combinations thereof.

[0021] The mutated nucleic acid sequence encoding mycobacterialisocitrate lyase provided by the present invention may also be obtainedfrom a library of mutants wherein the mutated mycobacteria are generatedusing methods of mutation which include, but are not limited to,signature-tagged mutagenesis, transposon mutagenesis, targeted genedisruption, illegitimate recombination and chemical mutagenesis. Thedisruption of a nucleic acid encoding isocitrate lyase may be performed,for example, by allelic exchange.

[0022] The mutant nucleic acid sequences encoding mycobacterialisocitrate lyase of the present invention may be prepared in severalways. For example, they can be prepared by isolating the nucleic acidsequences from a natural source, or by synthesis using recombinant DNAtechniques. In addition, mutated nucleic acid sequences encodingmycobacterial isocitrate lyase can be prepared using site mutagenesistechniques.

[0023] The present invention also provides a vector which comprises thenucleic acid encoding mycobacterial isocitrate lyase of the presentinvention, and a vector which comprises the mutated nucleic acidencoding mycobacterial isocitrate lyase of the present invention. Suchvectors may be constructed by inserting the nucleic acid encodingmycobacterial isocitrate lyase, or the mutated nucleic acid encodingmycobacterial isocitrate lyase into a suitable vector. The term“inserted” as used herein means the ligation of a foreign DNA fragmentand vector DNA by techniques such as the annealing of compatiblecohesive ends generated by restriction endonuclease digestion or by useof blunt end ligation techniques. Other methods of ligating DNAmolecules will be apparent to one skilled in the art.

[0024] Vectors may be derived from a number of different sources. Theycan be plasmids, viral-derived nucleic acids, lytic bacteriophagederived from phage lambda (λ), cosmids or filamentous single-strandedbacteriophages such as M13. Depending upon the type of host cell intowhich the vector is introduced, vectors may be bacterial or eukaryotic.Bacterial vectors are derived from many sources including the genomes ofplasmids and phage. Eukaryotic vectors are also constructed from anumber of different sources, e.g. yeast plasmids and viruses. Somevectors, called shuttle vectors, are capable of replicating in bothbacteria and eukaryotes. The nucleic acid from which the vector isderived is usually greatly reduced in size so that only those genesessential for its autonomous replication remain. The reduction in sizeenables the vectors to accommodate large segments of foreign DNA.Examples of suitable vectors into which the nucleic acid encodingmycobacterial isocitrate lyase or the mutated nucleic acid encodingmycobacterial isocitrate lyase can be inserted include but are notlimited to the shuttle vector pYUB412, shuttle vector pMP7, pJM056,pBR322, pUC18, pUC19, pHSV-106, pJS97, pJS98, M13mp18, M13mp19, pSPORT1, pGem, pSPORT 2, pSVSPORT 1, pBluescript II, λZapII, λgt10, λgt11,λgt22A, and λZIPLOX. Other suitable vectors are obvious to one skilledin the art.

[0025] The vector of the present invention may be introduced into a hostcell and may exist in integrated or unintegrated form within the hostcell. When in unintegrated form, the vector is capable of autonomousreplication. The term “host cell” as used herein means the bacterial oreukaryotic cell into which the vector is introduced. As used herein,“introduced” is a general term indicating that one of a variety of meanshas been used to allow the vector to enter the intracellular environmentof the host cell in such a way that it exists in stable and expressibleform therein.

[0026] Some bacterial and eukaryotic vectors have been engineered sothat they are capable of expressing inserted nucleic acids to highlevels within the host cell. Such vectors utilize one of a number ofpowerful promoters to direct the high level of expression. For example,in vectors for the expression of a gene in a bacterial host cell such asE. coli, the lac operator-promoter or the tac promoter are often used.Eukaryotic vectors use promoter-enhancer sequences of viral genes,especially those of tumor viruses. Expression can be controlled in bothbacterial and eukaryotic cells using inducible promoters such as the lacoperator-promoter in E. coli or metallothionine or mouse mammary tumorvirus promoters in eukaryotic cells. As used herein, “expression” refersto the ability of the vector to transcribe the inserted nucleic acidinto mRNA so that synthesis of the protein encoded by the insertednucleic acid can occur.

[0027] Vectors may be introduced into host cells by a number oftechniques known to those skilled in the art, e.g. electroporation, DEAEdextran, cationic liposome fusion, protoplast fusion, DNAcoated-microprojectile bombardment, and infection with recombinantreplication-defective retroviruses. The term “transformation” denotesthe introduction of a vector into a bacterial or eukaryotic host cell.As such, it encompasses transformation of bacterial cells andtransfection, transduction and related methods in eukaryotic cells.

[0028] Any one of a number of suitable bacterial or eukaryotic hostcells may be transformed with the vector of the present invention.Examples of suitable host cells are known to one skilled in the art andinclude but are not limited to mycobacterial cells such as M.tuburculosis, M, smegmatis, M. avium, M. kansasii, M. zenopi, M. simiae,M. gastri, M. szulgai, M. gordonae, M. chelonea, M. leprae, M.bovis-BCG, M. intracellulare, M. habana, M. lufu, M. phlei, M.fortuitum, M. paratuburculosis and M. scrofulaceum, and bacterial cellssuch as E.coli strains c600, c600hfl, HB101, LE392, Y1090, JM103, JM109,JM101, JM107, Y1088, Y1089, Y1090, Y1090(ZZ), DM1, PH10B, DH11S, DH125,RR1, TB1 and SURE, Bacillus subtilis, Agrobacterium tumefaciens,Bacillus megaterium; and eukaryotic cells such as Pichia pastoris,Chlamydomonas reinhardtii, Cryptococcus neoformans, Neurospora crassa,Podospora anserina, Saccharomyces cerevisiae, Saccharomyces pombe,Uncinula necator, cultured insect cells, cultured chicken fibroblasts,cultured hamster cells, cultured human cells such as HT1080, MCF7, 143Band cultured mouse cells such as EL4 and NIH3T3 cells.

[0029] The present invention also provides a purified and isolatedmycobacterial isocitrate lyase protein and analogues thereof, andincludes mycobacterial isocitrate lyase protein isolated from nature andmycobacterial isocitrate lyase protein which is recombinantly produced.As used herein “analogues” may be any protein having the same action asisocitrate lyase.

[0030] The isocitrate lyase protein provided by the present inventionmay be isolated from any species of mycobacteria, including, but notlimited to, M. tuburculosis, M, smegmatis, M. avium, M. kansasii, M.zenopi, M. simiae, M. gastri, M. szulgai, M. gordonae, M. chelonea, M.leprae, M. bovis-BCG, M. intracellulare, M. habana, M. lufu, M. phlei,M. fortuitum, M. paratuburculosis and M. scrofulaceum. In a preferredembodiment of the invention, the mycobacterial isocitrate lyase isisolated from M. tuberculosis. In another embodiment of the invention,mycobacterial isocitrate lyase is isolated from M. smegmatis.

[0031] In a preferred embodiment of the invention, the isocitrate lyaseprotein is M. tuberculosis isocitrate lyase and has the amino acidsequence containing in FIG. 1. In another embodiment of the invention,the isocitrate lyase protein is M. smegmatis isocitrate lyase and hasthe amino acid sequence containing in FIG. 2.

[0032] The present invention further provides for proteins encoded bymutated nucleic acids encoding mycobacterial isocitrate lyase. Themutation in the nucleic acid encoding the protein of the presentinvention may be generated in said nucleic acid using methods known toone of skill in the art. Such methods of mutation include, but are notlimited to, signature-tagged mutagenesis, transposon mutagenesis,targeted gene disruption, illegitimate recombination and chemicalmutagenesis. In a preferred embodiment of the invention, the isocitratelyase protein is encoded by a mutated M. tuburculosis nucleic acid. In amore preferred embodiment on the invention, the mutation in the M.tuburculosis nucleic acid encoding isocitrate lyase is generated bydisruption. Disruption of a nucleic acid encoding isocitrate lyase maybe performed, for example, by allelic exchange. It is to be understoodthat the present invention also provides for nucleic acid sequenceswherein any or all of the above described mutations coexist in thenucleic acid encoding mycobacterial isocitrate lyase in any combinationsthereof.

[0033] The isocitrate protein of the present may also be encoded by amutated nucleic acid sequence obtained from a library of mutants whereinthe mutated mycobacteria are generated using methods of mutation whichinclude, but are not limited to, signature-tagged mutagenesis,transposon mutagenesis, targeted gene disruption, illegitimaterecombination and chemical mutagenesis. The disruption of a nucleic acidencoding isocitrate lyase may be performed, for example, by allelicexchange.

[0034] The isocitrate proteins and the amino acid sequences of theseproteins may be isolated from mycobacteria such as M. tuburculosis, M,smegmatis, M. avium, M. kansasii, M. zenopi, M. simiae, M. gastri, M.szulgai, M. gordonae, M. chelonea, M. leprae, M. bovis-BCG, M.intracellulare, M. habana, M. lufu, M. phlei, M. fortuitum, M.paratuburculosis and M. scrofulaceum. The isocitrate proteins of thepresent invention and the amino acid sequences of these proteins also besynthesized by methods commonly known to one skilled in the art (ModernTechniques of Peptide and Amino Acid Analysis, John Wiley & Sons (1981);M. Bodansky, Principles of Peptide Synthesis, Springer Verlag (1984)).Examples of methods that may be employed in the synthesis of the aminoacids sequences, and mutants of these sequences include, but are notlimited to, solid phase peptide synthesis, solution method peptidesynthesis, and synthesis using any of the commercially available peptidesynthesizers. The amino acid sequences, and mutants thereof, may containcoupling agents and protecting groups used in the synthesis of theprotein sequences, and are well known to one of skill in the art.

[0035] The present invention also provides a host cell transformed witha vector encoding mycobacterial isocitrate lyase. The introduction ofthe recombinant vector containing the DNA sequence into the cell may beeffected by methods known to one skilled in the art, such aselectroporation, DEAE Dextran, cationic liposome fusion, protoplastfusion, DNA coated microprojectile bombardment, injection withrecombinant replication-defective viruses, homologous recombination, andnaked DNA transfer. It will be appreciated by those skilled in the artthat any of the above methods of DNA transfer may be combined.

[0036] The present invention also provides for antibodies immunoreactivewith mycobacterial isocitrate lyase and analogues thereof. Theantibodies of the present invention include antibodies immunoreactivewith non-functional mycobacterial isocitrate lyase, i.e., isocitratelyase which is inactive or exhibits only reduced activity in vivo. Thenon-functional isocitrate lyase recognized by the antibodies of thepresent invention may result from one or more mutations in the nucleicacid encoding mycobacterial isocitrate lyase or from one or moredeficiencies in the cell s protein synthesis and maturation pathwayswhich result in a mycobacterial isocitrate lyase with altered secondaryor tertiary structure.

[0037] The antibodies of the present invention may be monoclonal orpolyclonal and are produced by techniques well known to those skilled inthe art, e.g., polyclonal antibody can be produced by immunizing arabbit, mouse, or rat with purified mycobacterial isocitrate lyase andmonoclonal antibody may be produced by removing the spleen from theimmunized rabbit, mouse or rat and fusing the spleen cells with myelomacells to form a hybridoma which, when grown in culture, will produce amonoclonal antibody. Labeling of the antibodies of the present inventionmay be accomplished by standard techniques using one of the variety ofdifferent chemiluminescent and radioactive labels known in the art. Theantibodies of the present invention may also be incorporated into kitswhich include an appropriate labeling system, buffers and othernecessary reagents for use in a variety of detection and diagnosticapplications.

[0038] Further provided by the present invention is a mycobacterium thatcontains a mutation in its isocitrate lyase gene. The mycobacterium maybe, for example, M. tuburculosis, M, smegmatis, M. avium, M. kansasii,M. zenopi, M. simiae, M. gastri, M. szulgai, M. gordonae, M. chelonea,M. leprae, M. bovis-BCG, M. intracellulare, M. habana, M. lufu, M.phlei, M. fortuitum, M. paratuburculosis or M. scrofulaceum.

[0039] The mutation may be generated in the isocitrate gene of themycobacterium using methods known to one of skill in the art. Suchmethods of mutation include, but are not limited to, transposonmutagenesis, targeted gene disruption, illegitimate recombination andchemical mutagenesis. In a preferred embodiment of the invention, themycobacterium is M. tuburculosis. In a more preferred embodiment of theinvention, the mutation in the M. tuburculosis nucleic acid encodingisocitrate lyase is generated by disruption. Disruption of a nucleicacid encoding isocitrate lyase may be performed, for example, by allelicexchange. It is to be understood that the present invention alsoprovides for nucleic acid sequences wherein any or all of the abovedescribed mutations coexist in the nucleic acid encoding mycobacterialisocitrate lyase in any combinations thereof.

[0040] The mycobacterium having a mutated isocitrate lyase gene providedby the present invention may also be obtained from a library of mutantswherein the mutated mycobacteria are generated using methods of mutationwhich include, but are not limited to, transposon mutagenesis, targetedgene disruption, illegitimate recombination and chemical mutagenesis.The disruption of a nucleic acid encoding isocitrate lyase may beperformed, for example, by allelic exchange.

[0041] The present invention also provides an agent that inhibits theactivity or expression of a mycobacterial lyase protein. The inventorshave shown that the isocitrate lyase gene of M. tuberculosis is requiredfor stationary-phase persistence of M. tuberculosis. Agents that inhibitthe activity or expression of the mycobacterial lyase protein wouldspecifically kill stationary phase M. tuberculosis in vivo. By attackingstationary phase ‘persisters’, an isocitrate lyase inhibitor couldsignificantly accelerate the eradication of infection with chemotherapy.

[0042] Further provided by the present invention is a method ofproducing a compound that inhibits isocitrate lyase activity comprising:(a) providing purified isocitrate lyase; (b) determining the molecularstructure of said isocitrate lyase; (c) locating the binding sites ofsaid isocitrate lyase; (d) creating a compound with a similar structureto a binding site; (e) determining that said compound inhibits thebiochemical activity of isocitrate lyase. The design and synthesis of anisocitrate lyase inhibitor should be relatively simple for tworeasons: 1) the enzyme is a small protein of just 428 amino acids; 2)the ICL substrate (isocitrate) is a small molecule of known molecularstructure. Isocitrate lyase is a particularly attractive target forrational drug design because this enzyme is not found in human cells;therefore, an isocitrate lyase inhibitor would not be expected todisplay toxicity for human cells. Because the isocitrate lyase substrateis a small molecule, a specific inhibitor might be obtained byhigh-throughput screening of a small-molecule library using purifiedisocitrate lyase enzyme. This approach would be facilitated by the factthat a simple in vitro assay for isocitrate lyase activity alreadyexists. Alternatively, structure-based rational design of an isocitratelyase inhibitor would ideally proceed by the following steps: 1) The M.tuberculosis enzyme isocitrate lyase would be overproduced inEscherichia coli and purified; 2) The 3-dimensional structure of thepurified isocitrate lyase protein bound to its substrate (isocitrate)would be obtained by X-ray crystallography; 3) knowledge of the3-dimensional co-crystal structure would permit chemical modificationsof the substrate to be made in such a way that the modified substratewould act as a ‘pseudo-substrate’. Binding of the ‘pseudo-substrate’ tothe isocitrate lyase enzyme would sterically hinder binding of the bonafide substrate (isocitrate) and would therefore inhibit isocitrate lyasefunction. It might also be possible to design isocitrate lyaseinhibitors that would become covalently linked to the isocitrate lyaseenzyme, resulting in irreversible inhibition.

[0043] Also provided by the present invention is a method of determiningwhether a drug is effective against Mycobacterium tuberculosiscomprising (a) providing isolated isocitrate lyase; (b) providing acandidate drug; (c) mixing isocitrate lyase with substrates theglyoxylate shunt in the presence or absence of the candidate drug; and(d) measuring any inhibition of biosynthesis of malate caused by thepresence of the drug.

[0044] Further provided by the present invention is a method fortreating or preventing tuberculosis in a subject comprisingadministering an effective amount of an agent that inhibits the activityor expression of mycobacterial isocitrate lyase protein to treat thetuberculosis.

[0045] Finally, the present invention provides a method of identifying agene required for persistence of Mycobacteria tuburculosis in a subjectcomprising: (a) obtaining a library of M. tuberculosis mutants; (b)screening said library for an inactivated gene; (c) infecting a mammalwith M. tuberculosis containing the inactivated gene; (d) determiningwhether there is persistence of the M. tuberculosis containing theinactivated gene in said mammal, said absence of persistence indicatingthat the inactive gene is necessary for persistence of M. tuberculosis.

[0046] Examples of mycobacterial genes which may be analyzed using thismethod of identifying genes required for persistence include, but arenot limited to, the malate synthase gene, and genes involved in fattyacid catabolism.

[0047] The mutation in the mycobacterial gene may be generated usingmethods known to one of skill in the art. Such methods of mutationinclude, but are not limited to, signature-tagged mutations, transposonmutagenesis, targeted gene disruption, illegitimate recombination andchemical mutagenesis. In a preferred embodiment of the invention, themycobacterium is M. tuburculosis. In a more preferred embodiment of theinvention, the mutation in M. tuburculosis is in the isocitrate lyasegene, and is mutated by disruption. Disruption of an M. tuburculosisgene may be performed, for example, by allelic exchange.

[0048] The mycobacterial library containing the mutations is thenscreened using a phenotypic analysis.

[0049] The present invention is described in the following ExperimentalDetails Section which is set forth to aid in the understanding of theinvention, and should not be construed to limit in any way the inventionas defined in the claims which follow thereafter.

Experimental Details Section

[0050] I. Materials and Methods

[0051] Mycobacterial strains and growth conditions. mc²155 is an“efficient plasmid transformation” (ept) mutant of Mycobacteriumsmegmatis (Snapper et al., 1990). Virulent Mycobacterium tuberculosisstrain Erdman (Trudeau Institute, saranac Lake, N.Y.) was passagedthrough mice, grown once in culture, washed twice with phosphatebuffered saline containing 0.1% Tween-80 and 10% glycerol, and stored inaliquots at 80° C.

[0052] Liquid growth medium was Middlebrook 7H9 broth (4.7 g/L 7H9 broth(DifCo), 0.2% dextrose, 0.5% glycerol, 0.5% bovine serum albumin (BSA)Fraction V (Boehringer Mannheim Biochemicals), 15 mM NaCl, 0.1%Tween-80). Solid growth medium for M. smegmatis was Middlebrook 7H10agar (19 g/L 7H10 agar (DifCo), 0.2% dextrose, 0.5% glycerol, 0.5% BSA,15 mM NaCl). Solid growth medium for M. tuberculosis was Middlebrook7H10 oleic acid agar (19 g/L 7H10 agar (DifCo), 0.5% glycerol, 10%Middlebrook OADC Enrichment (BBL)). For analysis of M. smegmatis growthon alternative carbon sources, solid growth medium was MycobacterialBasal (MB) medium (1.5% Bacto agar (DifCo), 0.5 mM CaCl₂, 0.5 mM MgCl₂,0.1% KH₂PO₄, 0.25% Na₂HPO₄, 0.5% NH₄Cl, 0.2% K₂SO₄, 0.08 mg/L ZnCl₂, 0.4mg/L FeCl₃.6H₂O, 0.02 mg/L CuCl₂.2H₂O, 0.02 mg/L MnCl₂.4H₂O, 0.02 mg/LNa₂B₄O₇.10H₂O, 0.02 mg/L (NH₄)6Mo₇O24.4H₂O) supplemented with either0.5% glucose or 0.5% sodium acetate. For growth of M. tuberculosis, MBmedium was supplemented with 0.5% BSA. Inclusion of BSA alone did notsupport growth of M. tuberculosis in the absence of an added carbonsource (data not shown) . All mycobacterial media contained 100 μg/mLcycloheximide to prevent fungal contamination. Where noted, 50 μg/mLhygromycin (Boehringer Mannheim Biochemicals) or 30 μg/mL kanamycin(Sigma) was included.

[0053] For assessment of survival during anaerobiosis, 3.5 liter DifCoanaerobic jars and anaerobic system envelopes were used as per themanufacturer's instructions. Mycobacteria were pre-adapted toanaerobiosis essentially as described previously (Wayne, 1982). Briefly,mycobacterial cells were inoculated into Middlebrook 7H9 broth atapproximately 1×10⁸ CFU per mL, aliquoted in 15 mL plastic screwcaptubes (Corning), and incubated at 37° C. upright without agitation for 6weeks before use.

[0054] Ethyl Methane Sulfonate (EMS) mutagenesis. M. smegmatis strain mc²155 bacilli were grown to mid-log phase (A600 0.5-1.0) in Middlebrook7H9 broth and harvested by centrifugation. Cells were washed twice andresuspended in an equal volume of 0.1 M phosphate buffer (pH 7.0)containing 0.1% Tween-80. Ethyl methane sulfonate (EMS) (Sigma) wasadded to 2.5% and cells were incubated at 37° C. for 75-90 minutes,resulting in ˜25% cell survival. Mutagenesis was terminated by theaddition of thiosulfate (Sigma) to 4%. Cells were washed once with 4%thiosulfate containing 0.1% Tween-80, washed twice with Middlebrook 7H9broth, and resuspended in Middlebrook 7H9 broth. After 3 hours' recoveryat 37° C., the mutagenized cell suspension was sonicated and passedthrough a 5.0 micron pore syringe-filter (Micron Separations, Inc.) toremove clumps. Filtered cells were diluted and plated on Middlebrook7H10 agar supplemented with 5 g/L Casamino acids (Sigma), 0.1 g/LDL-a,e-diaminopimelic acid (DAP) (Sigma), and 0.02 g/L tryptophan(Sigma) to permit recovery of amino acid auxotrophs. Colonies werepatched to fresh plates to form an ordered collection of mutants andscreened for the presence of auxotrophs to assess the complexity of themutant bank. Out of 6000 individual colonies analyzed, 136 amino acidauxotrophs were recovered (frequency 2.3%), representing most of theclasses of mutants expected.

[0055] Isolation and complementation of Ace⁻ mutants of M. smegmatis.The EMS mutant bank was screened for isolates unable to grow on MBmedium containing acetate as sole carbon source (Ace⁻ phenotype). ElevenAce⁻ mutants were recovered from 3000 picks screened (frequency 0.37%).To identify potential isocitrate lyase (icl) mutants among these, eachof the eleven Ace⁻ mutants was transformed with a plasmid expressing theEscherichia coli icl gene from the mycobacterial hsp60 heat shockpromoter (pJM007, see “Plasmids”) and screened for growth on MB+acetatemedium. Two of the eleven Ace⁻ mutants (ACE1023 and ACE2832) werecomplemented by E. coli icl (frequency 0.067%).

[0056] The mycobacterial icl genes were isolated from cosmid librariesconsisting of large (20-40 kbp) fragments of genomic DNA from M.smegmatis or M. tuberculosis inserted into the cosmid vector pYUB412.pYUB412 is an E.coli-Mycobacterium shuttle vector containing the oriEreplication origin for maintenance in E. coli, the mycobacteriophage L5attachment/integration system for site-specific insertion into themycobacterial genome (Lee et al. 1991), the hygromycinphosphotransferase gene conferring hygromycin resistance inmycobacteria, the b-lactamase gene conferring ampicillin resistance inE. coli, and dual cos sites for packaging in phage l heads. The pYUB412polylinker is flanked by recognition sites for restriction endonucleasePacI (New England Biolabs), which has few or no recognition sites inmycobacterial genomic DNA; digestion with PacI therefore releases theintact genomic insert. Construction of the mycobacterial genomiclibraries (generously provided to us by F.-C. Bange) will be describedelsewhere. The libraries were electroporated into the Ace⁻ strainACE1023 and Ace+ transformants were selected on MB+acetate medium.

[0057] Integrated cosmid inserts were recovered from the mycobacterialgenomic DNA as follows. pYUB412 cosmid arms were prepared by digestionwith XbaI (New England Biolabs) to separate cos sites, dephosphorylationwith calf intestinal phosphatase (Boehringer Mannheim Biochemicals) toprevent self-ligation, and digestion with PacI. Genomic DNA was preparedfrom individual Ace+ transformants as described (Mizuguchi and Tokunaga1970) and digested with PacI. pYUB412 cosmid arms and PacI-digestedgenomic DNA were ligated (DNA ligase from Boehringer MannheimBiochemicals), packaged into phage l heads using GigaPack Gold(Stratagene) packaging mix, and transduced into E. coli strain STBL2(Stratagene), all according to the manufacturers' instructions.Transductants were selected on LB medium containing 50 μg/mL ampicillin.Plasmid DNA was isolated from individual transductants using standardmethods and electroporated into ACE1023. Transformants were selected onMiddlebrook 7H10 agar containing 50 μg/mL hygromycin and screened forthe ability to grow on MB+dextrose and MB+acetate.

[0058] Southern blot analysis. Genomic and plasmid DNAs were digestedwith restriction endonucleases (New England Biolabs) as indicated in thetext and separated on 1.0% agarose-TBE gels. Gels were processed andtransferred to Hybond-N+ nylon membranes (Amersham) as per themanufacturer's instructions. The M. tuberculosis icl probe fragment wasprepared by polymerase chain reaction (PCR) amplification of a 981 bpSacII fragment subcloned into the vector pKS+ (Stratagene), using T3 andT7 oligonucleotides flanking the polylinker site and Vent polymerase(New England Biolabs). The amplified fragment was labeled using theEnhanced Chemiluminescence kit (Amersham) and the blot was probed anddeveloped according to the manufacturer's instructions.

[0059] Analysis of mycobacterial growth and persistence in mice. MaleC57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, Me.).Female 129SvEv mice were obtained from Taconic (Germantown, N.Y.). TheB6X129 F1 progeny of the C57BL/6J×129SvEv cross were used forexperiments. Frozen stocks of wild-type (icl+) and icl⁻ M. tuberculosisstrain Erdman were prepared by growing cells to mid-log phase (A6000.5-1.0) in Middlebrook 7H9 broth, washing cells twice withphosphate-buffered saline containing 0.1% Tween-80 and 10% glycerol, andstoring in aliquots at −80ÿC. Aliquots were thawed, diluted asappropriate in phosphate-buffered saline containing 0.1% Tween-80, andsonicated in two 10 sec bursts using a cup-horn sonicator. Mice wereinfected intravenously by injection into a lateral tail vein ofapproximately 1×10⁶ CFU of tubercle bacilli in a volume of 0.1 mL.

[0060] At timepoints indicated in the text, mice (four per group) weresacrificed by cervical dislocation and organs were removed aseptically.Organs were transferred to plastic Stomacher bags (Tekmar, Cincinnati,Ohio) with phosphate buffered saline containing 0.1% Tween-80 andhomogenized using a Stomacher homogenizer (Tekmar). Organ homogenateswere diluted in phosphate buffered saline containing 0.1% Tween-80 andplated on Middlebrook 7H10 oleic acid agar. Colonies were scored after3-4 weeks' incubation at 37° C.

[0061] II. Results

[0062] The Krebs cycle serves dual functions in metabolism: generationof metabolic energy by oxidation of acetyl CoA, and provision ofintermediates for several essential biosynthetic pathways (FIG. 1).Sustained operation of the Krebs cycle therefore requires an anapleroticfunction to replenish intermediates that are siphoned off forbiosyntheses. Pyruvate carboxylase satisfies this requirement for cellsgrowing on carbohydrates (FIG. 1). This pathway is not operative whencells are grown on C2 carbon sources such as acetate or fatty acids,since carbon from these substrates enters metabolism at the level ofacetyl CoA (FIG. 1). Instead, a novel anaplerotic pathway, theglyoxylate shunt, is induced during growth on C2 substrates. Theglyoxylate shunt consists of two enzymes, isocitrate lyase and malatesynthase, which catalyze the formation of one molecule of malate (aKrebs cycle intermediate) from two molecules of acetyl CoA (FIG. 1).Synthesis of these enzymes is repressed during growth on carbohydrates.The glyoxylate shunt is present in many eubacterial species and in somesimple eukaryotes (including fungi) but is absent in vertebrates.

[0063] Metabolic studies of tubercle bacilli purified directly from thelungs of chronically infected mice suggested that fatty acids may serveas an important source of carbon and energy for mycobacteria within theinfected host. If so, then the fatty acid b-oxidation pathway and theglyoxylate shunt may be essential for in vivo growth or persistence. Theinventors have begun to address this hypothesis using newly-developedmolecular genetic techniques for the generation of targeted mutations inmycobacteria, focusing first on the enzymes of the glyoxylate shunt.Here, we describe the isolation of the genes encoding isocitrate lyasein fast- and slow-growing mycobacteria, targeted disruption of the icllocus in virulent M. tuberculosis, and phenotypic analysis of the M.tuberculosis icl mutant.

[0064] Isolation of Ace mutants of Mycobacterium smegmatis. In order toidentify functions required for utilization of C₂ carbon sources inmycobacteria, a genetic screen was conducted in the fast-growing speciesMycobacterium smegmatis. A library of mutant clones was generated bymutagenesis with ethane methyl sulfonate (EMS), as described inMaterials and Methods. From a collection of 3000 mutant clones, 11mutants (frequency 0.37%) were identified that were incapable of growthon acetate as sole carbon source (Ace⁻ phenotype). To identify potentialisocitrate lyase (icl) mutants, the 11 Ace mutants were transformed withan E. coli-Mycobacterium shuttle plasmid expressing the E. coli icl genefrom the mycobacterial hsp60 promoter. Growth of two of the 11 Acemutants on acetate (frequency 0.067%) was restored by expression of E.coli icl. One of these mutants (ACE1023) displayed a tight Ace-phenotype(FIG. 2A, E) and a low reversion rate (<10⁻⁷; data not shown) and wasselected for further analysis.

[0065] Complementation of a putative icl mutant of M. smegmatis withgenomic libraries of M. smegmatis and M. tuberculosis. The ACE1023mutant of M. smegmatis was transformed with genomic cosmid librariescontaining inserts of M. smegmatis or M. tuberculosis genomic DNA. Theselibraries were constructed in the shuttle vector pYUB412, which utilizesthe mycobacteriophage L5 attachment/integration system for single-copyinsertion into the attB site of the mycobacterial chromosome.Transformants were selected on 7H10+AD medium containing 50 μg/mLhygromycin and screened for complementation of the Ace phenotype. Growthon acetate was restored in approximately 1 of 250 transformants obtainedwith the M. smegmatis library and in approximately 1 of 200transformants obtained with the M. tuberculosis library. In order toensure that growth on acetate resulted from expression of thecomplementing clone and not from reversion of the mutation, theintegrated plasmid inserts were retrieved (see Materials and Methods)and retransformed into the ACE1023 strain. Twelve independent clonesfrom the M. smegmatis library and 11 independent clones from the M.tuberculosis library were analyzed; all restored growth on acetate whenretransformed into ACE1023. One clone from each library was arbitrarilyselected for further analysis. These cosmid clones contained inserts of20-40 kbp. By a combination of subcloning and complementation analysis,smaller complementing fragments were obtained: a 2558 base-pairHpaI-EcoRI genomic fragment from M. smegmatis and a 2674 base-pairBamHI-ClaI genomic fragment from M. tuberculosis (FIG. 2E-H, 3A).

[0066] Nucleotide sequence and Southern blot analysis of the M.smegmatis and M. tuberculosis genes encoding isocitrate lyase (ICL). Thenucleotide sequences of the putative icl loci from M. smegmatis and M.tuberculosis (see previous section) were determined and potential openreading frames (ORFs) were identified. Each fragment contained an ORFencoding a conceptual protein homologous to the isocitrate lyaseproteins of other gram-positive and gram-negative organisms. Theconceptual ICL proteins from M. smegmatis and M. tuberculosis are 92%identical to each other and both are ˜84% identical to the ICL proteinfrom Rhodococcus fasciens (FIG. 3B).

[0067] Situated just downstream of the icl genes in both M. smegmatisand M. tuberculosis are ORFs with significant homology to genes encoding3-hydroxybutyryl-CoA dehydrogenase (BHBD) in other eubacterial species(FIG. 3A, C). The putative BHBD proteins encoded by M. smegmatis and M.tuberculosis are 83% identical to each other and both are 45% identicalto the BHBD protein from Clostridium acetobutylicum. In the latterspecies, BHBD catalyzes the conversion of acetoacetyl-CoA to3-hydroxybutyryl-CoA in the butyrate/butanol fermentation pathway forgeneration of ATP and regeneration of oxidized NAD+ during anaerobicgrowth. Mycobacteria, however, are obligate aerobes and are not capableof growing anaerobically by fermentation. The possible significance of aBHBD homolog in mycobacteria will be discussed later.

[0068] The identity of the cloned icl genes was confirmed by Southernblot analysis of M. smegmatis and M. tuberculosis genomic DNAs usingfragments derived from the cloned icl genes as probes.

[0069] Targeted disruption of the isocitrate lyase gene in virulent M.tuberculosis. The icl gene was disrupted in the virulent Erdman strainof M. tuberculosis using an efficient method for allelic exchange. Thismethod employs the counter-selectable marker sacB, which is lethal inthe presence of sucrose. Successful application of sacB for efficientallelic exchange in M. tuberculosis was described recently by Pelicic etal. (1997). A 685 base-pair XhoI fragment internal to the M.tuberculosis icl gene was replaced with the hygromycinphosphotransferase (hpt) gene from Streptomyces hygroscopicus (FIG. 5A).The recombinant icl::hpt allele was incapable of rescuing growth of theACE1023 mutant on acetate, confirming that the disrupted gene was notfunctional (data not shown). The icl::hpt cassette was inserted into theshuttle vector pMP7, which contains the oriE and oriM replicationorigins for plasmid maintenance in E. coli and mycobacteria(respectively), the aph gene conferring kanamycin resistance, and thecounter-selectable sacB marker from Bacillus subtilis. The resultingplasmid (pJM056) was electroporated into M. tuberculosis strain ErdmanMC²3030 and transformants were selected on solid medium containing 50μg/mL hygromycin. MC²3030, containing the icl::hpt recombinant allellehas been deposited under the terms of the Budapest Treaty on ______ withthe American Type Culture Collection (ATCC), Rockville, Md., andassigned ATCC Accession No. ______. In the absence of antibioticselection, the plasmid was rapidly lost in broth cultures (data notshown). Therefore, transformants grown in the presence of hygromycinwere expected to lose the plasmid following allelic exchange between thechromsomal icl gene and the icl::hpt allele on the plasmid.

[0070] Five individual colonies obtained from independenttransformations with pJM056 were picked, inoculated separately intoliquid medium containing 50 μg/mL hygromycin, and grown to saturation toallow time for recombination to occur between the plasmid and thebacterial chromosome. The saturated broth cultures were diluted andplated on solid medium containing 50 μg/mL hygromycin +/−5% sucrose.Expression of sacB in M. tuberculosis was lethal on solid mediumcontaining 5% sucrose, permitting selection against cells that retainedthe pMP7 vector. The relative plating efficiencies of the individualbroth cultures on medium plus/minus sucrose were variable (10⁰ to 10⁻³),suggesting that loss of sacB function occurred at different times in thegrowth of the cultures. Twenty-five hgmr sucr colonies derived from eachliquid culture were screened for the ability to grow on kanamycin. Twoof five cultures yielded colonies that were uniformly (25/25) resistantto kanamycin; these presumably carried mutations in sacB and werediscarded. The three remaining cultures yielded hgmr sucr colonies thatwere sensitive (25/25) to kanamycin. From each of these cultures,individual hgmr sucr kans colonies were expanded and analyzed bySouthern blot (FIG. 5B). Of the 10 colonies analyzed, three coloniesobtained from two independent cultures contained only the disruptedicl::hpt allele, establishing that allelic exchange had occurred (FIG.5B).

[0071] Phenotypic analysis of the M. tuberculosis isocitrate lyasemutant. As expected, disruption of icl abrogated growth on solid mediumcontaining acetate as sole carbon source (data not shown). In contrast,growth of the M. tuberculosis icl⁻ mutant was normal when glucose andglycerol were provided as carbon sources (FIG. 6A). Suryanarayana et al.(1973) demonstrated that ICL levels increased during entry of M.tuberculosis into stationary phase, suggesting that the glyoxylate shuntmight play a role in stasis survival. They postulated that endogenousfatty acids might serve as an alternative carbon source for maintenancemetabolism following depletion of exogenous carbon sources. However, wefound that the ability of the icl⁻ mutant to survive long-term stasiswas unimpaired (FIG. 6A).

[0072] Wayne and Lin (1982) found that ICL levels increased duringadaptation of tubercle bacilli to anaerobiosis. They proposed theexistence of a novel pathway involving ICL and another enzyme, glycinedehydrogenase, for the regeneration of NAD+ from NADH at oxygen tensionstoo low to support respiration. However, the demonstration that survivalof oxygen starvation was not impaired by disruption of icl (FIG. 6B)suggests that this pathway, if it exists, is not essential foradaptation to anaerobiosis.

[0073] The aim of the studies described herein was to determine whetherthe glyoxylate shunt is important for in vivo nutrition of tuberclebacilli. The ability of the M. tuberculosis icl⁻ mutant to grow andpersist in the mouse model of tuberculosis was therefore assessed. Micewere infected by the intravenous route with approximately 2×10⁶colony-forming units (CFU) of either wild-type (icl+) M. tuberculosis orthe icl− mutant. Bacterial loads in the lungs were determined at 1 dayand at 1, 2, 4, 8, 12, and 16 weeks post-infection (FIG. 7A). In theearly phase of infection (up to 2 weeks), prior to the emergence ofadaptive immunity (Orme, 1994), in vivo growth of the icl− and wild-type(icl+) bacilli was similar. Following the emergence of bacteriostaticimmunity after 2 weeks, growth of wild-type M. tuberculosis ceased and aconstant bacterial load was maintained thereafter. In contrast, from 2weeks onwards, the titer of the icl mutant in the lungs fell steadily,resulting in a 40-fold reduction in the bacterial burden by 16 weekspost-infection. These results demonstrate that isocitrate lyase is notrequired for early growth of M. tuberculosis prior to the emergence ofbacteriostatic immunity, but is important for chronic persistence oncegrowth ceases. The “persistence defect” of the icl mutant resulted in astriking attenuation of disease progression (FIG. 7B).

[0074] III. Discussion

[0075] It is a truism that M. tuberculosis must acquire nutrients fromthe infected host in order to replicate and cause disease. Little isknown, however, of the mechanisms that are involved in nutrientacquisition in vivo. A number of potential sources of carbon and energyare abundant in mammalian cells, but it is not known which of thesesubstrates are available to mycobacteria growing within the confines oftightly-apposed vacuolar membranes. Tubercle bacilli may modify thevacuolar membrane in order to gain access to the rich variety ofsubstrates that are abundant in the cytoplasm of the host cell. However,the inability of a leucine auxotroph of M. bovis BCG to replicate withinmacrophages suggests that access to cytoplasmic constituents may belimited. One substrate that would be readily accessible to mycobacteriagrowing within the parasitophorous vacuole is the fatty acids of thevacuolar membrane itself. In mammalian cells, fatty acids arepotentially one of the most abundant carbon substrates available(Wheeler and Ratledge, 1994). M. tuberculosis produces a number oflipases and phospholipases capable of liberating free fatty acids frommembrane-associated and storage forms such as phospholipids andtriglycerides. Continuous fusion of the mycobacterium-containing vacuolewith host-derived vesicles could serve to replenish membrane consumed bythe parasite. M. tuberculosis also encodes the molecular machineryrequired for utilization of fatty acids as sole carbon source: theb-oxidation pathway for breakdown of fatty acids to assimilableacetyl-CoA units, and the glyoxylate shunt required for replenishment ofKrebs cycle intermediates depleted by biosynthetic pathways. The enzymesof both pathways are expressed by pathogenic mycobacteria growing invivo. Metabolic studies of in vivo grown mycobacteria also suggestedthat fatty acids may serve as a major source of carbon and energy duringgrowth within the infected host.

References

[0076] Artman, M., and Bekierkunst, A. (1960) Studies on Mycobacteriumtuberculosis H37Rv grown in vivo: utilization of glucose. Proc. Soc.Exp. Biol. Med. 105: 609-612.

[0077] Bange, F.-C., Brown, A. M., and Jacobs, W. R., Jr. (1996) Leucineauxotrophy restricts growth of Mycobacterium bovis BCG in macrophages.Infect. Immun. 64: 1794-1799.

[0078] Clark, D. P. and Cronan, J. E., Jr. (1996) Two-carbon compoundsand fatty acids as carbon sources. In Escherichia coli and Salmonella:Cellular and Molecular Biology. Neidhardt, F. C. (ed.). Washington,D.C.: ASM Press, pp. 343-357.

[0079] Clemens, D. L. (1996) Characterization of the Mycobacteriumtuberculosis phagosome. Trends Microbiol. 4: 113-118.

[0080] Cronan, J. E., Jr. and LaPorte, D. (1996) Tricarboxylic acidcycle and glyoxylate bypass. In Escherichia coli and Salmonella:Cellular and Molecular Biology. Neidhardt, F. C. (ed.). Washington,D.C.: ASM Press, pp. 206-216.

[0081] Heifets, L. B. and Good, R. C. (1994) Current laboratory methodsfor the diagnosis of tuberculosis. In Tuberculosis: Pathogenesis,Protection, and Control. Bloom, B. R. (ed.). Washington, D.C.: ASMPress, pp. 85-110.

[0082] Lee, M. H., Pascopella, L., Jacobs, W. R., Jr., and Hatfull, G.F. (1991) Site-specific integration of mycobacteriophage L5:Integration-proficient vectors for Mycobacterium smegmatis,Mycobacterium tuberculosis, and bacille Calmette-Guérin. Proc. Natl.Acad. Sci. USA 88: 3111-3115.

[0083] McAdam, R. A., Weisbrod, T. R., Martin, J., Scuderi, J. D.,Brown, A. M., Cirillo, J. D., Bloom, B. R., and Jacobs, W. R., Jr.(1995) In vivo growth characteristics of leucine and methionineauxotrophic mutants of Mycobacterium bovis BCG generated by transposonmutagenesis. Infect. Immun. 63: 1004-1012.

[0084] Medlar, E. M. (1948) The pathogenesis of minimal pulmonarytuberculosis: a study of 1,225 necropsies in cases of unexpected andsudden death. Am. Rev. Tuberc. 58: 583-611.

[0085] Mizuguchi, Y., and Tokunaga, T. (1970) Method for isolation ofdeoxyribonucleic acid from mycobacteria. J. Bacteriol. 104: 1020-1021.

[0086] Moreira, A. L., Wang, J., Tsenova-Berkova, L., Hellmann, W.,Freedman, V. H., and Kaplan, G. (1997) Sequestration of Mycobacteriumtuberculosis in tight vacuoles in vivo in lung macrophages of miceinfected by the respiratory route. Infect. Immun. 65: 305-308.

[0087] Pelicic, V., Jackson, M., Reyrat, J.-M., Jacobs, W. R., Jr.,Gicquel, B., and Guilhot, C. (1997) Efficient allelic exchange andtransposon mutagenesis in Mycobacterium tuberculosis. Proc. Natl. Acad.Sci. USA 94: 10955-10960.

[0088] Ratledge, C. (1976) The physiology of the mycobacteria. InAdvances in Microbial Physiology. Rose, A. H., and Tempest, D. W. (ed.).New York: Academic Press, pp. 115-244.

[0089] Segal, W., and Bloch, H. (1956) Biochemical differentiation ofMycobacterium tuberculosis grown in vivo and in vitro. J. Bacteriol. 72:132-141.

[0090] Segal, W. (1984) Growth dynamics of in vivo and in vitro grownmycobacterial pathogens. In The Mycobacteria: A Sourcebook. Kubica, G.P. and Wayne, L. G. (ed.). New York, N.Y.: Marcel Dekker Inc., pp.547-573.

[0091] Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T., andJacobs, W. R., Jr. (1990) Isolation and characterization of efficientplasmid transformation mutants of Mycobacterium smegmatis. Mol.Microbiol. 4: 1911-1919.

[0092] Sturgill-Koszycki, S., Schaible, U. E., and Russell, D. G. (1996)Mycobacterium-containing phagosomes are accessible to early endosomesand reflect a transitional state in normal phagosome biogenesis. EMBO J.15: 6960-6968.

[0093] Suryanarayana Murthy, P., Sirsi, M., and Ramakrishnan, T. (1973)Effect of age on the enzymes of tricarboxylic acid and related cycles inMycobacterium tuberculosis H37Rv. Am. Rev. Respir. Dis. 108: 689-690.

[0094] Wayne, L. G., and Lin, K.-Y. (1982) Glyoxylate metabolism andadaptation of Mycobacterium tuberculosis to survival under anaerobicconditions. Infect. Immun. 37: 1042-1049.

[0095] Wayne, L. G. (1994) Cultivation of Mycobacterium tuberculosis forresearch purposes. In Tuberculosis: Pathogenesis, Protection, andControl. Bloom, B. R. (ed.). Washington, D.C.: ASM Press, pp. 73-83.

[0096] Wheeler, P. R., and Ratledge, C. (1988) Use of carbon sources forlipid biosynthesis in Mycobacterium leprae: a comparison with otherpathogenic mycobacteria. J. Gen. Microbiol. 134: 2111-2121.

[0097] Wheeler, P. R. and Ratledge, C. (1994) Metabolism ofMycobacterium tuberculosis. In Tuberculosis: Pathogenesis, Protection,and Control. Bloom, B. R. (ed.). Washington, D.C.: ASM Press, pp.353-385.

What is claimed is:
 1. A purified and isolated nucleic acid encodingmycobacterial isocitrate lyase.
 2. The nucleic acid of claim 1 obtainedfrom Mycobacterium smegmatis.
 3. The nucleic acid of claim 1 obtainedfrom Mycobacterium tuberculosis.
 4. The nucleic acid sequence of claim 2having the nucleotide sequence contained in FIG.
 1. 5. The nucleic acidsequence of claim 3 having the nucleotide sequence contained in FIG. 2.6. A purified and isolated mutated mycobacterial isocitrate lyasenucleic acid.
 7. The nucleic acid of claim 6 obtained from Mycobacteriumsmegmatis.
 8. The nucleic acid of claim 7 which has one or moredisruption, deletion, insertion, point, substitution, nonsense, misense,polymorphism or rearrangement mutation.
 9. The nucleic acid of claim 6obtained from Mycobacterium tuberculosis.
 10. The nucleic acid of claim9 which has one or more disruption, deletion, insertion, point,substitution, nonsense, misense, polymorphism or rearrangement mutation.13. A recombinant vector comprising nucleic acid that encodesmycobacterial isocitrate lyase.
 14. The recombinant vector of claim 13wherein the nucleic acid encoding isocitrate lyase is obtained fromMycobacterium smegmatis.
 15. The recombinant vector of claim 13 whereinthe nucleic acid encoding isocitrate lyase is obtained fromMycobacterium tuberculosis.
 16. The recombinant vector of claim 14wherein the nucleic acid encoding isocitrate lyase has the nucleotidesequence contained in FIG.
 1. 17. The recombinant vector of claim 15wherein the nucleic acid encoding isocitrate lyase has the nucleotidesequence contained in FIG.
 2. 18. A recombinant vector comprisingmutated nucleic acid that encodes mycobacterial isocitrate lyase. 19.The recombinant vector of claim 18 wherein the mutated nucleic acidencoding isocitrate lyase is obtained from Mycobacterium smegmatis. 20.The recombinant vector of claim 19 wherein the mutated nucleic acidencoding isocitrate lyase has one or more disruption, deletion,insertion, point, substitution, nonsense, misense, polymorphism orrearrangement mutation.
 21. The recombinant vector of claim 18 whereinthe mutated nucleic acid encoding isocitrate lyase is obtained fromMycobacterium tuberculosis.
 22. The recombinant vector of claim 21wherein the mutated nucleic acid has one or more disruption, deletion,insertion, point, substitution, nonsense, misense, polymorphism orrearrangement mutation.
 23. A mycobacterium that contains a mutation itsisocitrate lyase gene.
 24. The mycobacterium of claim 23 which isMycobacterium tuberculosis.
 25. The mycobacterium of claim 24, whereinthe mutation is a disruption, deletion, insertion, point, substitution,nonsense, misense, polymorphism or rearrangement mutation.
 26. Themycobacterium of claim 25, wherein the mutation is a disruptionmutation.
 27. The mycobacterium of claim 26, deposited under ATCCAccession No. ______.
 28. The mycobacterium of claim 23 which isMycobacterium smegmatis.
 29. The mycobacterium of claim 28, wherein themutation is a disruption, deletion, insertion, point, substitution,nonsense, misense, polymorphism or rearrangement mutation.
 30. Apurified, active protein encoded by a mycobacterial isocitrate lyasegene having the amino acid sequence contained in FIG.
 1. 31. A purified,active protein encoded by a mycobacterial isocitrate lyase gene havingthe amino acid sequence contained in FIG.
 2. 32. A purified proteinencoded by a mutated isocitrate lyase nucleic acid.
 33. The protein ofclaim 32, wherein the mutated mycobacterial isocitrate lyase nucleicacid has one or more disruption, deletion, insertion, point,substitution, nonsense, misense, polymorphism or rearrangementmutations.
 34. An agent that inhibits the activity or expression of amycobacterial isocitrate lyase protein.
 35. A method for treating orpreventing tuberculosis in a subject comprising administering aneffective amount of agent of claim 34 to the subject to treat thetuberculosis.
 36. A method of determining whether a drug is effectiveagainst Mycobacterium tuberculosis comprising: (a) providing isolatedisocitrate lyase; (b) providing a candidate drug; (c) mixing isocitratelyase with substrates for the glyoxylate shunt in the presence orabsence of the candidate drug; and (d) measuring any inhibition ofbiosynthesis of malate caused by the presence of the drug.
 37. A drugidentified by the method of claim
 36. 38. A method of producing acompound that inhibits isocitrate lyase activity comprising: (a)providing purified isocitrate lyase; (b) determining the molecularstructure of said isocitrate lyase; (c) locating the binding sites ofsaid isocitrate lyase; (d) creating a compound with a similar structureto a binding site; (e) determining that said compound inhibits thebiochemical activity of isocitrate lyase.
 39. A method of identifying agene required for persistence of Mycobacteria tuburculosis in a subjectcomprising: (a) obtaining a library of M. tuberculosis mutants; (b)screening said library for an inactivated gene; (c) infecting a mammalwith M. tuberculosis containing the inactivated gene; (d) determiningwhether there is persistence of the M. tuberculosis containing theinactivated gene in said mammal, said absence of persistence indicatingthat the inactive gene is necessary for persistence of M. tuberculosis.40. The method of claim 39, wherein the gene is a gene involved in fattyacid catabolism.
 41. The method of claim 39, wherein the gene is themalate synthase gene.
 42. The method of claim 39, wherein the library ofM. tuberculosis mutants is generated by signature-tagged mutagenesis,transposon mutagenesis, targeted gene disruption, illegitimaterecombination or chemical mutagenesis.